The overall goal of this murine gastric acid aspiration model is to produce a sterile acute lung injury in a mouse that has close fidelity to the pathogenesis of acid pneumonitis observed in humans. This method can help answer key questions in the critical care setting about identifying therapeutic strategies that can lessen the severity and improve the outcome of sterile acute lung injury. The main advantage of this technique is that it produces a clinically relevant model that can take advantage of the numerous genetically engineered animals available for dissecting out the involved mechanisms.
Demonstrating the procedure will be Dr.Bruce Davidson, a faculty colleague and collaborator, and Barbara Mullen, our senior trade research associate. Induce anesthesia in a chamber with 2.75%isoflurane. Confirm anesthesia has been achieved by a lack of response to a toe pinch and applying ointment to the animals eyes.
Use an electric trimmer to shave the ventral portion of the neck and disinfect the surgical area. Then, use a 15 centimeter length of one zero braided silk suture to suspend the mouse by the superior incisors in the supine position on a 60 degree incline dissection board, with the nose inserted in a nose cone, delivering 2.75%isoflurane. Further disinfect the surgical area by applying povidone iodine.
Remove the excess antiseptic with gauze. Infiltrate the midline of the neck from the sternal notch to the inferior section of the jaw with 100 microliters of 0.5%bupivacaine. Next, use a scalpel to make a 1-1.5 centimeter longitudinal midline incision in the neck followed by blunt dissection of the fascial membrane between the salivary glands with two curved serrated forceps.
When the trachea is visible, use the forceps to pull one side of the paratracheal musculature aside. While dissecting between the muscle fibers longitudinally next to the trachea. Once the dissection plane is established, keep the paratracheal muscle retracted with one pair of forceps, while working under the trachea with the second forceps.
Then, use the forceps to place a second 15 centimeter strand of one zero braided silk suture under the trachea. For the intra-tracheal instillation, first fill a 0.5 milileter syringe equipped with a 22 gauge needle with 0.2 milliliters of air followed by 3.6 milliliters per kilogram of hydrochloric acid. Immediately pull the tracheal suture superiorly to provide traction and have an assistant gently squeeze the ribcage to expel the vital capacity.
Insert the syringe needle, bevel side up, into the trachea between the first and second cartilaginous rings below the larynx. Then, when the bevel is just past the trachea, with the needle as parallel to the trachea as possible, release the chest and quickly administer the hydrochloric acid bolus to assure the dispersion and deposition of the treatment into the distal airway and alveoli. Remove the needle after the first spontaneous breath to confirm inhalation of the entire bolus volume into the lungs, as incomplete delivery leads to upper airway injury only, and failing to represent the severe pneumonitis acute lung injury observed in human patients.
After the first spontaneous breath, use surgical staples to close the incision, and use a five milliliter syringe with a 26 gauge needle to inject one milliliter of sterile normal saline subcutaneously into the scruff of the neck for fluid resuscitation. After confirming a lack of response to a toe pinch, secure the animal in the supine position as just demonstrated, and apply eye ointment. After reopening the staples, make a midline longitudinal incision through the skin from the lower abdominal wall through the previous neck incision to the mandible.
Followed by another midline incision through the peritoneal musculature. Use sponges and retract the abdominal organs to expose the retroperitoneal space. Then, clamp the left external iliac artery vein to provide retraction force and use forceps to gently remove the fascia covering the abdominal aorta and vena cava.
Use a one CC syringe, equipped with a 26 gauge by half inch needle, to collect blood from the aorta. Followed by perforation of the diaphragm to cause lung collapse. Use bone cutting scissors to cut away the diaphragm.
Then cut through the ribcage parallel to and on both sides of the sternum, and use a locking hemostat to retract the sternal flap. Inject five milliliters of 37 degrees Celsius HBSS with calcium and magnesium into the right ventricle to flush the lung vasculature. Then, incline the dissection board to a 60 degree angle.
Facilitate cannula insertion by piercing the ventral side of the trachea just below the larynx using a 20 gauge needle. And then insert a 20 gauge by half inch stainless steel cannula into the trachea through the tracheal aperture as just demonstrated. Securing the cannula with a one zero suture.
When the cannula is in place, fill a syringe with five milliliters of sterile normal saline. Screw the syringe into one port of a three way stopcock. Attach an empty five milliliter syringe to a second port, and attach the last port to the intratracheal cannula.
Then, instill one milliliter of normal saline into the lungs, and recover the lavage with a second syringe. Twenty four hours post lung injury as just demonstrated, necrotic cells, loss of lung parenchymal architecture, and cells and debris within the airspaces with significant polymorphonuclear neutrophil infiltration are clearly observed. Similar to that observed in human ARDS patients histologically.
The albumin concentration in the bronchoalveal lavage is increased following acid aspiration compared to normal saline controls at two and five hours post injury. The albumin concentration is significantly higher at five hours post acid injury compared to two hours, due to the inflammatory response. This is consistent with the pathophysiology observed in human patients following acid aspiration.
Once mastered, the acute lung injury technique can be completed in three to five minutes. And the specimen procurement following the various times post injury can be completed in six to eight minutes if the methods are performed properly. While attempting this procedure, it is important not to damage the trachea and esophagus during the blunt dissection.
After it's development, this technique paved the way for researchers in the field of acute lung injury to explore therapeutic strategies for decreasing the severity of pulmonary injury due to a major inducement of sterile acute inflammatory lung injury. Employing this procedure, the murine model of acid pneumonitis can also be used to examine the interaction of low PH insults, with other associated pulmonary insults, including food particles and bacteria. After watching this video, you should have a good understanding of how to produce a sterile acute lung injury in mice that closely resembles the pathogensis of acid pneumonitis observed in humans.
Don't forget that a good scavenging system should be employed when working with the volatile anesthetics used to perform this procedure.
